The present invention is related to the construction and application of recombinant vectors containing DNA sequences for encoding, and efficient expression of, enzymatically active cytochromes P450 in mammalian cells. The invention also is related to immortalized human bronchial epithelial cells containing various cytochrome P450 genes and the uses of these cells.
The cytochromes P450 are a large family of hemoprotein enzymes capable of metabolizing xenobiotics such as drugs, carcinogens and environmental pollutants as well as endobiotics such as steriods, fatty acids and prostaglandins. Some members of the cytochrome P450 family are inducible in both animals and cultured cells, while other constitutive forms are non-inducible. This group of enzymes carry out beneficial metabolic activities by detoxification of xenobiotics as well as harmful metabolic conversion of xenobiotics to toxic, mutagenic and carcinogenic forms (Gelboin, Physiol. Rev., 60:1107-1166, 1980).
In animals, multiple molecular forms of cytochrome P450s are expressed simultaneously and they all exhibit common physical and biological properties. The multiplicity and common properties of cytochromes P450 make it difficult to separate their different forms, especially the minor forms. Even in situations where P450 cytochromes have been isolated in purified form by conventional enzyme purification procedures, they have been removed from the natural biological membrane association and therefore require the addition of NADPH-cytochrome P450 reductase and other cell fractions for enzymatic activity. These additional factors have prevented a clearer understanding of the role and function of the individual cytochrome forms in metabolism, detoxification, and activation of both xenobiotic and endobiotic substrates.
Toxicological testing of drugs, potential carcinogens, food products, food additives and food contaminants has been performed in animals and more recently in in vitro systems, such as bacteria (Ames test) and animal cell culture models. These systems are disadvantaged since they do not have human-specific metabolism. Therefore, extrapolation to determine the human risk is difficult and potentially inaccurate. The bacterial test systems and some of the animal cell culture models lack complete metabolic activity and would not detect any harmful compounds which depend upon activation by metabolic pathways, for example, by the cytochrome P450 enzymes. In the past this situation was circumvented by adding metabolizing enzyme isolated from rat livers to the cultured animal cells. This approach poses two significant problems. First, the resulting metabolism is not necessarily the same as in man. Secondly, highly-reactive metabolites might not reach their target molecule and, consequently, escape detection.
Although human metabolizing enzymes have been introduced into a human cell line, this system suffers from serious deficiencies. (Crespi, Progress in Clinical and Biological Research, Vol. 340B Mendelsohn and Albertini (eds) Wiley-Liss, New York 97-106, 1990.) The human cells are lymphoblasts which do not constitute a major target tissue of cytotoxins, mutagens, or carcinogens and have no natural cytochrome P450 activity in the absence of inducers. In addition, other enzymes involved in the activation process, for example, epoxide hydrolase, are missing in these cells and must be introduced by gene transfer methodology. This system therefore comprises an artificial model with a questionable correlation to the in vivo situation.